Gsk3 ligands and polynucleotides encoding gsk3 ligands

ABSTRACT

The invention relates to kinase ligands and polyligands. In particular, the invention relates to ligands and polyligands that modulate GSK3 activity. The ligands and polyligands are utilized as research tools or as therapeutics. The invention includes linkage of the ligands and polyligands to a cellular localization signal, epitope tag and/or a reporter. The invention also includes polynucleotides encoding the ligands and polyligands.

This Application claims benefit of priority to U.S. 60/865,587, filed 13Nov. 2006.

FIELD OF INVENTION

The invention relates to mammalian kinase ligands, substrates andmodulators. In particular, the invention relates to polypeptides,polypeptide compositions and polynucleotides that encode polypeptidesthat are ligands, substrates, and/or modulators of GSK3. The inventionalso relates to polyligands that are homopolyligands orheteropolyligands that modulate GSK3 activity. The invention alsorelates to ligands and polyligands tethered to a subcellular location.

This application has subject matter related to application Ser. Nos.10/724,532 (now U.S. Pat. No. 7,071,295), 10/682,764 (US2004/0185556,PCT/US2004/013517, WO2005/040336), 11/233,246, and US20040572011P(WO2005116231), PCT/U506/60065, PCT/US06/60062. Each of these patentsand applications is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Kinases are enzymes that catalyze the addition of phosphate to amolecule. The addition of phosphate by a kinase is calledphosphorylation. When the kinase substrate is a protein molecule, theamino acids commonly phosphorylated are serine, threonine and tyrosine.Phosphatases are enzymes that remove phosphate from a molecule. Theremoval of phosphate is called dephosphorylation. Kinases andphosphatases often represent competing forces within a cell to transmit,attenuate, or otherwise modulate cellular signals and cellular controlmechanisms. Kinases and phosphatases have both overlapping and uniquenatural substrates. Cellular signals and control mechanisms, asregulated by kinases, phosphatases, and their natural substrates are atarget of research tool design and drug design.

Mammalian Gycogen Synthase Kinase-3 is also known as GSK3. GSK3, whichhas alpha and beta isoforms, can phosphorylate serine and threonineresidues in protein or peptide substrates. GSK3 also autophosphorylatesitself on a tyrosine residue. Some, but not all, in vivo GSK3phosphorylation events depend upon prior phosphorylation of thesubstrate by another cellular kinase. The enzymatic activity, activationand regulation of GSK3 have been studied. Many cellular substrates ofGSK3 have been identified. Furthermore, polypeptides have been studiedto examine GSK3 substrate specificity. While polypeptides and variantsthereof have been studied as individual GSK3 substrates or ligands,mixed ligands linked together as polyligands that modulate GSK3 activityhave not been demonstrated before this invention. An aspect of theinvention is to provide novel, modular, inhibitors of GSK3 activity bymodifying one or more natural substrates either by truncation or byamino acid substitution. A further aspect of the invention is thesubcellular localization of a GSK3 inhibitor, ligand, or polyligand bylinking to a subcellular localization signal.

Examples of GSK3 substrates and regulators include those described inBax, et al. 2001 Structure 9:1143-52, Beals, et al. 1997 Science275:1930-4, Casaday, et al. 2004 J Virol 78:13501-11, Chu, et al. 1996 JBiol Chem 271:30847-57, Cole, et al. 2004 Biochem J 377:249-55, Fiol, etal. 1990 J Biol Chem 265:6061-5, Frame, et al. 2001 Biochem J 359:1-16,Fujimuro, et al. 2005 J Virol 79:10429-41, Godemann, et al. 1999 FEBSLett 454:157-64, Hughes, et al. 1992 Biochem J 288 (Pt 1):309-14,Kirschenbaum, et al. 2001 J Biol Chem 276:7366-75, Liberman, et al. 2005J Biol Chem 280:4422-8, Litovchick, et al. 2004 Mol Cell Biol24:8970-80, Liu, et al. 2006 BMC Mol Biol 7:14, Medunjanin, et al. 2005J Biol Chem 280:33006-14, Nikolakaki, et al. 1993 Oncogene 8:833-40,Piwien-Pilipuk, et al. 2001 J Biol Chem 276:19664-71, Plotkin, et al.2003 J Pharmacol Exp Ther 305:974-80, Rhoads 1999 J Biol Chem274:30337-40, Rubinfeld, et al. 1996 Science 272:1023-6, Ryan, et al.2005 J Cell Biol 171:327-35, Sato, et al. 2002 J Biol Chem 277:42060-5,Sheorain, et al. 1985 J Biol Chem 260:12287-92, Takeda, et al. 2000 HumMol Genet 9:125-32, Trivedi, et al. 2005 J Cell Sci 118:993-1005, andWelsh, et al. 1997 Anal Biochem 244: 16-21.

Design and synthesis of polypeptide ligands that modulatecalcium/calmodulin-dependent protein kinase and that localize to thecardiac sarco(endo)plasmic reticulum was performed by Ji et al. (J BiolChem (2003) 278:25063-71). Ji et al. accomplished this by generatingexpression constructs that localized calcium/calmodulin-dependentprotein kinase inhibitory polypeptide ligands to the sarcoplasmicreticulum by fusing a sarcoplasmic reticulum localization signal derivedfrom phospholamban to a polypeptide ligand. See also U.S. Pat. No.7,071,295.

DETAILED DESCRIPTION OF POLYPEPTIDE AND POLYNUCLEOTIDE SEQUENCES

SEQ ID NOS: 1-12 are example polyligands and polynucleotides encodingthem.

Specifically, the GSK3 polyligand of SEQ ID NO:1 is encoded by SEQ IDNO:2 and by SEQ ID NO:3, wherein the codons of have been optimized formammalian expression. SEQ ID NO:4 includes flanking restriction sites.SEQ ID NO: 1 is an embodiment of a polyligand of the structure X-X,wherein X is SEQ ID NO: 119. A polyligand of structure X-X is alsocalled herein a homopolyligand, shown generically in FIG. 1A.

SEQ ID NO:4 is an embodiment of a polyligand of the structure A-S1-B,wherein A is SEQ ID NO: 117 and B is SEQ ID NO: 118, and wherein S1 is aspacer of amino acid sequence GGAPAGG. The GSK3 polyligand of SEQ IDNO:4 is encoded by SEQ ID NO:5 and SEQ ID NO:6, wherein the codons ofhave been optimized for mammalian expression. SEQ ID NO:6 includesflanking restriction sites. A polyligand of structure A-S1-B is alsocalled herein a heteropolyligand, shown generically in FIG. 4A.

SEQ ID NO:7 is an embodiment of a polyligand of the structureX-S2-Y-S3-Z, wherein X is SEQ ID NO:120, Y is SEQ ID NO:121, Z is SEQ IDNO:116, and wherein S2 is a five amino acid spacer with the sequenceGGAGG and S3 is a four amino acid spacer with the sequence GGGG. TheGSK3 polyligand of SEQ ID NO:7 is encoded by SEQ ID NO:8 and by SEQ IDNO:9, wherein the codons have been optimized for mammalian expression.SEQ ID NO:9 includes flanking restriction sites. A polyligand ofstructure X-S2-Y-S3-Z is also called herein a heteropolyligand, showngenerically in FIG. 4B.

SEQ ID NO:10 is an embodiment of a polyligand of the structureX-S4-Y-S5-Z, wherein X is SEQ ID NO:48, Y is SEQ ID NO:49, Z is SEQ IDNO:50, wherein Xaa is alanine, and wherein S4 is a four amino acidspacer with the sequence AAAA and S5 is an four amino acid spacer withthe sequence GGAA. The GSK3 polyligand of SEQ ID NO:10 is encoded by SEQID NO:11, SEQ ID NO: 12, wherein the codons have been optimized formammalian expression. SEQ ID NO: 12 includes flanking restriction sites.A polyligand of structure X-S4-Y-S5-Z is also called herein aheteropolyligand, shown generically in FIG. 4B.

SEQ ID NOS:13-43 are full length GSK3 protein substrates. Thesesequences have the following public database accession numbers:AAB69872, AAB29227, Q13144, AAH06195, AAK61224, CAA79497, NP_(—)000029,P15941, NP_(—)444284, NP_(—)002219, CAA38500, NP_(—)034722, CAA25015,AAD00450, AAC50235, NP_(—)034013, NP_(—)005185, AAQ24858, NP_(—)032627,NP_(—)937820, Q00613, P10636, P46821, NP_(—)000012, NP_(—)000116,AAA85777, NP_(—)060082, NP_(—)005535, NP_(—)034700, NP_(—)001377,AAD46501, NP 063937, NP_(—)002084, NP_(—)005470. Each of the sequencesrepresented by these accession numbers is incorporated by referenceherein. In SEQ ID NOS: 13-43, the positions of the amino acid(s)phosphorylatable by GSK3 are represented by Xaa. In wild-type proteins,Xaa is serine or threonine. In the ligands of the invention, Xaa is anyamino acid.

SEQ ID NO:44 and SEQ ID NO:45 are full length GSK3 alpha and betapolypeptide sequences. In SEQ ID NOS:44-45, the positions of the aminoacid(s) autophosphorylatable by GSK3 are represented by Xaa. Inwild-type GSK3, Xaa is tyrosine. In the ligands of the invention, Xaa isany amino acid.

SEQ ID NOS:46-111 are peptide sequences including subsequences of SEQ IDNOS:13-45, which represent examples of peptide ligand sequences wherethe location of the GSK3 phosphorylatable amino acid in the naturalpolypeptide is designated as Xaa.

SEQ ID NOS:112-114 are peptide substrates, which represent examples ofpeptide ligand sequences where the location of the GSK3 phosphorylatableamino acid in the natural polypeptide is designated as Xaa.

SEQ ID NOS:115-122 are inhibitors of GSK3.

SEQ ID NOS:46-122 represent examples of monomeric peptide ligandsequences. Amino acid sequences containing Xaa encompass peptides whereXaa is any amino acid.

DETAILED DESCRIPTION OF DRAWINGS

FIGS. 1A-1C show examples of homopolymeric ligands without spacers.

FIGS. 2A-2C show examples of homopolymeric ligands with spacers.

FIGS. 3A-3E show examples of heteropolymeric ligands without spacers.

FIGS. 4A-4F show examples of heteropolymeric ligands with spacers.

FIGS. 5A-5G show examples of ligands and polymeric ligands linked to anoptional epitope tag.

FIGS. 6A-6G show examples of ligands and polymeric ligands linked to anoptional reporter.

FIGS. 7A-7G show examples of ligands and polymeric ligands linked to anoptional localization signal.

FIGS. 8A-8G show examples of ligands and polymeric ligands linked to anoptional localization signal and an optional epitope tag.

FIGS. 9A-9G show examples of gene constructs where ligands andpolyligands are linked to an optional localization signal, an optionalepitope tag, and an optional reporter.

FIGS. 10A-10D show examples of vectors containing ligand geneconstructs.

FIG. 11 shows an example of a sequential cloning process useful forcombinatorial synthesis of polyligands.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to polypeptide ligands and polyligands for GSK3.Various embodiments of the GSK3 ligands and polyligands are representedin SEQ ID NOS:1-122. More specifically, the invention relates toligands, homopolyligands, and heteropolyligands that comprise any one ormore of SEQ ID NOS:46-122. Additionally, the invention relates toligands and polyligands comprising one or more subsequences of SEQ IDNOS: 13-45 or any portion thereof. Furthermore, the invention relates topolyligands with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and99% sequence identity to a polyligand comprising one or more of SEQ IDNOS:46-122 or any portion thereof. Furthermore, the invention relates topolyligands with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and99% sequence identity to a polyligand comprising one or moresubsequences of SEQ ID NOS:13-45.

Polyligands, which can be homopolyligands or heteropolyligands, arechimeric ligands composed of two or more monomeric polypeptide ligands.An example of a monomeric ligand is the polypeptide represented by SEQID NO:57, wherein Xaa is any amino acid. SEQ ID NO:57 is a selectedsubsequence of wild-type full length SEQ ID NO: 14, wherein the aminoacid corresponding to Xaa in the wild-type sequence is a serine orthreonine phosphorylatable by GSK3. An example of a homopolyligand is apolypeptide comprising a dimer or multimer of SEQ ID NO:57, wherein Xaais any amino acid. An example of a heteropolyligand is a polypeptidecomprising SEQ ID NO:46 and one or more of SEQ ID NOS:47-122, whereinXaa is any amino acid. There are numerous ways to combine SEQ IDNOS:46-122 into homopolymeric or heteropolymeric ligands. Furthermore,there are numerous ways to combine additional subsequences of SEQ IDNOS: 13-45 with each other and with SEQ ID NOS:46-122 to make polymericligands.

The polyligands of the invention optionally comprise spacer amino acidsbetween monomers. SEQ ID NO:7 is an embodiment of a polyligand of thestructure X-S2-Y-S3-Z, wherein X is SEQ ID NO:120, Y is SEQ ID NO:121, Zis SEQ ID NO:116, and wherein S2 and S3 are spacers. This inventionintends to capture all combinations of homopolyligands andheteropolyligands without limitation to the examples given above orbelow. In this description, use of the term “ligand(s)” encompassesmonomeric ligands, polymeric ligands, homopolymeric ligands and/orheteropolymeric ligands.

A monomeric ligand is a polypeptide where at least a portion of thepolypeptide is capable of being recognized by GSK3. The portion of thepolypeptide capable of recognition is termed the recognition motif. Inthe present invention, recognition motifs can be natural or synthetic.Examples of recognition motifs are well known in the art and include,but are not limited to, naturally occurring GSK3 substrates andpseudosubstrate motifs.

A polymeric ligand comprises two or more monomeric ligands.

A homopolymeric ligand is a polymeric ligand where each of the monomericligands is identical in amino acid sequence, except that aphosphorylatable residue may be substituted or modified in one or moreof the monomeric ligands.

A heteropolymeric ligand is a polymeric ligand where some of themonomeric ligands does not have an identical amino acid sequence.

The ligands of the invention are optionally linked to additionalmolecules or amino acids that provide an epitope tag, a reporter, and/ora cellular localization signal. The cellular localization signal targetsthe ligands to a region of a cell. The epitope tag and/or reporterand/or localization signal may be the same molecule. The epitope tagand/or reporter and/or localization signal may also be differentmolecules.

The invention also encompasses polynucleotides comprising a nucleotidesequence encoding ligands, homopolyligands, and heteropolyligands. Thenucleic acids of the invention are optionally linked to additionalnucleotide sequences encoding polypeptides with additional features,such as an epitope tag, a reporter, and/or a cellular localizationsignal. The polynucleotides are optionally flanked by nucleotidesequences comprising restriction endonuclease sites and othernucleotides needed for restriction endonuclese activity. The flankingsequences optionally provide unique cloning sites within a vector andoptionally provide directionality of subsequence cloning. Further, thenucleic acids of the invention are optionally incorporated into vectorpolynucleotides. The ligands, polyligands, and polynucleotides of thisinvention have utility as research tools and/or therapeutics.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to ligands and polyligands that are GSK3modulators. Various embodiments of ligands and polyligands arerepresented in SEQ ID NOS:1-122. Polyligands are chimeric ligandscomprising two or more monomeric polypeptide ligands. An example of amonomeric ligand is the polypeptide represented by SEQ ID NO:51, whereinXaa is any amino acid.

SEQ ID NO:51 is a selected subsequence of wild-type full length SEQ IDNO: 13, wherein the amino acid corresponding to Xaa in the wild-typesequence is a serine or threonine phosphorylatable by GSK3. Anotherexample of a monomeric ligand is the polypeptide represented by SEQ IDNO: 122. Each of SEQ ID NOS:46-122 represents an individual polypeptideligand in monomeric form, wherein Xaa is any amino acid. SEQ IDNOS:46-111 are selected examples of subsequences of SEQ ID NOS:13-45,however, other subsequences of SEQ ID NOS:13-45 may also be utilized asmonomeric ligands. Monomeric ligand subsequences of SEQ ID NOS:13-45 maybe wild-type subsequences. Additionally, monomeric ligand subsequencesof SEQ ID NOS:13-45 may have the GSK3 phosphorylatable amino acidsreplaced by other amino acids. Furthermore, monomeric ligands andpolyligands may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% sequence identity to a ligand comprising an amino acid sequence inone or more of SEQ ID NOS:46-111. Furthermore, monomeric ligands andpolyligands may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%and 99% sequence identity to a subsequence of SEQ ID NOS:13-45.

An example of a homopolyligand is a polypeptide comprising a dimer ormultimer of SEQ ID NO:46, wherein Xaa is any amino acid. An example of aheteropolyligand is a polypeptide comprising SEQ ID NO:46 and one ormore of SEQ ID NOS:47-122, wherein Xaa is any amino acid. There arenumerous ways to combine SEQ ID NOS:46-122 into homopolymeric orheteropolymeric ligands. Furthermore, there are numerous ways to combineadditional subsequences of SEQ ID NOS:13-45 with each other and with SEQID NOS:46-122 to make polymeric ligands.

Polyligands may comprise any two or more of SEQ ID NOS:46-122, whereinXaa is any amino acid. A dimer or multimer of SEQ ID NO: 111 is anexample of a homopolyligand. An example of a heteropolyligand is apolypeptide comprising SEQ ID NO: 122 and one or more of SEQ IDNOS:46-121. There are numerous ways to combine SEQ ID NOS:46-122 intohomopolymeric or heteropolymeric ligands. SEQ ID NOS:46-111 are selectedexamples of subsequences of SEQ ID NOS:13-45, however, additionalsubsequences, wild-type or mutated, may be utilized to form polyligands.The instant invention is directed to all possible combinations ofhomopolyligands and heteropolyligands without limitation.

SEQ ID NOS:13-45 show proteins that contain at least one serine,threonine or tyrosine residue phosphorylatable by GSK3, the positions ofwhich are represented by Xaa. SEQ ID NOS:46-111 are subsequences of SEQID NOS:13-45 where, again, the locations of the GSK3 phosphorylatableresidues are represented by Xaa. In nature, Xaa is, generally speaking,serine, threonine or tyrosine. In one embodiment of the instantinvention, Xaa can be any amino acid. Ligands where Xaa is serine orthreonine or tyrosine can be used as part of a polyligand, however inone embodiment, at least one phosphorylatable serine, threonine ortyrosine is replaced with another amino acid, such as one of thenaturally occurring amino acids including, alanine, aspartate,asparagine, cysteine, glutamate, glutamine, phenylalanine, glycine,histidine, isoleucine, leucine, lysine, methionine, proline, arginine,valine, or tryptophan. The Xaa may also be a non-naturally occurringamino acid. In another embodiment, the GSK3 phosphorylatable aminoacid(s) are replaced by alanine. The ligands and polyligands of theinvention are designed to modulate the endogenous effects of one or moreisoforms of GSK3.

In general, ligand monomers based on natural GSK3 substrates are builtby isolating a putative GSK3 phosphorylation recognition motif in a GSK3substrate. Sometimes it is desirable to modify the phosphorylatableresidue to an amino acid that cannot be phosphorylated. Additionalmonomers include the GSK3 recognition motif as well as amino acidsadjacent and contiguous on either side of the GSK3 recognition motif.Monomeric ligands may therefore be any length provided the monomerincludes the GSK3 recognition motif. For example, the monomer maycomprise an GSK3 recognition motif and at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30-100 or more amino acids adjacent to the recognitionmotif.

For example, in one embodiment, the invention comprises an inhibitor ofGSK3 comprising at least one copy of a peptide selected from the groupconsisting of:

a) a peptide at least 80% identical to a peptide comprising amino acidresidues corresponding to amino acid residues 638-664 of SEQ ID NO: 13,wherein the amino acid residue corresponding to amino acid residue 641,645, 649 or 653 of SEQ ID NO:13 is an amino acid residue other thanserine or threonine;b) a peptide at least 80% identical to a peptide comprising amino acidresidues corresponding to amino acid residues 635-667 of SEQ ID NO: 13,wherein the amino acid residue corresponding to amino acid residue 641,645, 649 or 653 of SEQ ID NO:13 is an amino acid residue other thanserine or threonine;c) a peptide at least 80% identical to a peptide comprising amino acidresidues corresponding to amino acid residues 632-671 of SEQ ID NO: 13,wherein the amino acid residue corresponding to amino acid residue 641,645, 649 or 653 of SEQ ID NO:13 is an amino acid residue other thanserine or threonine; andd) a peptide at least 80% identical to a peptide comprising amino acidresidues corresponding to amino acid residues 628-676 of SEQ ID NO: 13,wherein the amino acid residue corresponding to amino acid residue 641,645, 649 or 653 of SEQ ID NO:13 is an amino acid residue other thanserine or threonine.

As used herein, the terms “correspond(s) to” and “corresponding to,” asthey relate to sequence alignment, are intended to mean enumeratedpositions within a reference protein, e.g., glycogen synthase (SEQ IDNO: 13), and those positions that align with the positions on thereference protein. Thus, when the amino acid sequence of a subjectpeptide is aligned with the amino acid sequence of a reference peptide,e.g., SEQ ID NO:13, the amino acids in the subject peptide sequence that“correspond to” certain enumerated positions of the reference peptidesequence are those that align with these positions of the referencepeptide sequence, but are not necessarily in these exact numericalpositions of the reference sequence. Methods for aligning sequences fordetermining corresponding amino acids between sequences are describedbelow.

Additional embodiments of the invention include monomers (as describedabove) based on any putative or real substrate for GSK3, such assubstrates identified by SEQ ID NOS:13-114. Furthermore, if thesubstrate has more than one recognition motif, then more than onemonomer may be identified therein.

Further embodiments of the invention include monomers based on GSK3inhibitors and regulators, such as those identified by SEQ ID NOS:115-122 and subsequences thereof.

Another embodiment of the invention is a nucleic acid moleculecomprising a polynucleotide sequence encoding at least one copy of aligand peptide.

Another embodiment of the invention is a nucleic acid molecule whereinthe polynucleotide sequence encodes one or more copies of one or morepeptide ligands.

Another embodiment of the invention is a nucleic acid molecule whereinthe polynucleotide sequence encodes at least a number of copies of thepeptide selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9 or10.

Another embodiment of the invention is a vector comprising a nucleicacid molecule encoding at least one copy of a ligand or polyligand.

Another embodiment of the invention is a recombinant host cellcomprising a vector comprising a nucleic acid molecule encoding at leastone copy of a ligand or polyligand.

Another embodiment of the invention is a method of inhibiting GSK3 in acell comprising transfecting a vector comprising a nucleic acid moleculeencoding at least one copy of a ligand or polyligand into a host celland culturing the transfected host cell under conditions suitable toproduce at least one copy of the ligand or polyligand.

The invention also relates to modified inhibitors that are at leastabout 80%, 85%, 90% 95%, 96%, 97%, 98% or 99% identical to a referenceinhibitor. A “modified inhibitor” is used to mean a peptide that can becreated by addition, deletion or substitution of one or more amino acidsin the primary structure (amino acid sequence) of a inhibitor protein orpolypeptide. A “modified recognition motif” is a naturally occurringGSK3 recognition motif that has been modified by addition, deletion, orsubstitution of one or more amino acids in the primary structure (aminoacid sequence) of the motif. For example, a modified GSK3 recognitionmotif may be a motif where the phosphorylatable amino acid has beenmodified to a non-phosphorylatable amino acid. The terms “protein” and“polypeptide” are used interchangeably herein. The reference inhibitoris not necessarily a wild-type protein or a portion thereof. Thus, thereference inhibitor may be a protein or peptide whose sequence waspreviously modified over a wild-type protein. The reference inhibitormay or may not be the wild-type protein from a particular organism.

A polypeptide having an amino acid sequence at least, for example, about95% “identical” to a reference an amino acid sequence is understood tomean that the amino acid sequence of the polypeptide is identical to thereference sequence except that the amino acid sequence may include up toabout five modifications per each 100 amino acids of the reference aminoacid sequence encoding the reference peptide. In other words, to obtaina peptide having an amino acid sequence at least about 95% identical toa reference amino acid sequence, up to about 5% of the amino acidresidues of the reference sequence may be deleted or substituted withanother amino acid or a number of amino acids up to about 5% of thetotal amino acids in the reference sequence may be inserted into thereference sequence. These modifications of the reference sequence mayoccur at the N-terminus or C-terminus positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among amino acids in the reference sequence or inone or more contiguous groups within the reference sequence.

As used herein, “identity” is a measure of the identity of nucleotidesequences or amino acid sequences compared to a reference nucleotide oramino acid sequence. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. (See, e.g.,Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York (1988); Biocomputing: Informatics And Genome Projects,Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey (1994); von Heinje, G., Sequence Analysis In MolecularBiology, Academic Press (1987); and Sequence Analysis Primer, Gribskov,M. and Devereux, J., eds., M Stockton Press, New York (1991)). Whilethere exist several methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math48:1073 (1988)). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego (1994) and Carillo, H. & Lipton, D., Siam J AppliedMath 48:1073 (1988). Computer programs may also contain methods andalgorithms that calculate identity and similarity. Examples of computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, GCG program package(Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)), BLASTP,ExPASy, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403(1990)) and FASTDB. Examples of methods to determine identity andsimilarity are discussed in Michaels, G. and Garian, R., CurrentProtocols in Protein Science, Vol 1, John Wiley & Sons, Inc. (2000),which is incorporated by reference. In one embodiment of the presentinvention, the algorithm used to determine identity between two or morepolypeptides is BLASTP.

In another embodiment of the present invention, the algorithm used todetermine identity between two or more polypeptides is FASTDB, which isbased upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990), incorporated by reference). In a FASTDB sequence alignment, thequery and subject sequences are amino sequences. The result of sequencealignment is in percent identity. Parameters that may be used in aFASTDB alignment of amino acid sequences to calculate percent identityinclude, but are not limited to: Matrix=PAM, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or thelength of the subject amino sequence, whichever is shorter.

If the subject sequence is shorter or longer than the query sequencebecause of N-terminus or C-terminus additions or deletions, not becauseof internal additions or deletions, a manual correction can be made,because the FASTDB program does not account for N-terminus andC-terminus truncations or additions of the subject sequence whencalculating percent identity. For subject sequences truncated at bothends, relative to the query sequence, the percent identity is correctedby calculating the number of amino acids of the query sequence that areN- and C-terminus to the reference sequence that are notmatched/aligned, as a percent of the total amino acids of the querysequence. The results of the FASTDB sequence alignment determinematching/alignment. The alignment percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score can be used for the purposes of determining howalignments “correspond” to each other, as well as percentage identity.Residues of the query (subject) sequences or the reference sequence thatextend past the N- or C-termini of the reference or subject sequence,respectively, may be considered for the purposes of manually adjustingthe percent identity score. That is, residues that are notmatched/aligned with the N- or C-termini of the comparison sequence maybe counted when manually adjusting the percent identity score oralignment numbering.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue reference sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the FASTDB alignment does not show a match/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 reference sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected.

The polyligands of the invention optionally comprise spacer amino acidsbefore, after or between monomers. The length and composition of thespacer may vary. An example of a spacer is glycine, alanine,polyglycine, or polyalanine. Specific examples of spacers used betweenmonomers in SEQ ID NO:10 are the four amino acids AAAA and GGAA. Spaceramino acids may be any amino acid and are not limited to alanine andglycine. SEQ ID NO:10, depicted generically in FIG. 4B, represents aspecific example of a polyligand of the structure X-S4-Y-S5-Z, whereinX, Y and Z are chosen from SEQ ID NOS:46-122, and wherein S4 and S5 arespacers. The instant invention is directed to all combinations ofhomopolyligands and heteropolyligands, with or without spacers, andwithout limitation to the examples given above or below.

The ligands and polyligands of the invention are optionally linked toadditional molecules or amino acids that provide an epitope tag, areporter, and/or localize the ligand to a region of a cell (See FIGS.5A-5G, FIGS. 6A-6G, FIGS. 7A-7G, and FIGS. 8A-8G). Non-limiting examplesof epitope tags are FLAG™ (Kodak; Rochester, N.Y.), HA (hemagluttinin),c-Myc and His6. Non-limiting examples of reporters are alkalinephosphatase, galactosidase, peroxidase, luciferase and green fluorescentprotein (GFP). Non-limiting examples of cellular localizations aresarcoplamic reticulum, endoplasmic reticulum, mitochondria, golgiapparatus, nucleus, plasma membrane, apical membrane, and basolateralmembrane. The epitopes, reporters and localization signals are given byway of example and without limitation. The epitope tag, reporter and/orlocalization signal may be the same molecule. The epitope tag, reporterand/or localization signal may also be different molecules.

Ligands and polyligands and optional amino acids linked thereto can besynthesized chemically or recombinantly using techniques known in theart. Chemical synthesis techniques include but are not limited topeptide synthesis which is often performed using an automated peptidesynthesizer. Pepetides can also be synthesized utilizing non-automatedpeptide synthesis methods known in the art. Recombinant techniquesinclude insertion of ligand-encoding nucleic acids into expressionvectors, wherein nucleic acid expression products are synthesized usingcellular factors and processes.

Linkage of a cellular localization signal, epitope tag, or reporter to aligand or polyligand can include covalent or enzymatic linkage to theligand. When the localization signal comprises material other than apolypeptide, such as a lipid or carbohydrate, a chemical reaction tolink molecules may be utilized. Additionally, non-standard amino acidsand amino acids modified with lipids, carbohydrates, phosphate or othermolecules may be used as precursors to peptide synthesis. The ligands ofthe invention have therapeutic utility with or without localizationsignals. However, ligands linked to localization signals have utility assubcellular tools or therapeutics. For example, ligands depictedgenerically in FIGS. 7A-7G represent ligands with utility as subcellulartools or therapeutics. GSK3 ligand-containing gene constructs are alsodelivered via gene therapy. FIGS. 10B and 10C depict embodiments of genetherapy vectors for delivering and controlling polypeptide expression invivo. Polynucleotide sequences linked to the gene construct in FIGS. 10Band 10C include genome integration domains to facilitate integration ofthe transgene into a viral genome and/or host genome.

FIG. 10A shows a vector containing a GSK3 ligand gene construct, whereinthe ligand gene construct is releasable from the vector as a unit usefulfor generating transgenic animals. For example, the ligand geneconstruct, or transgene, is released from the vector backbone byrestriction endonuclease digestion. The released transgene is theninjected into pronuclei of fertilized mouse eggs; or the transgene isused to transform embryonic stem cells. The vector containing a ligandgene construct of FIG. 10A is also useful for transient transfection ofthe trangene, wherein the promoter and codons of the transgene areoptimized for the host organism. The vector containing a ligand geneconstruct of FIG. 10A is also useful for recombinant expression ofpolypeptides in fermentable organisms adaptable for small or large scaleproduction, wherein the promoter and codons of the transgene areoptimized for the fermentation host organism.

FIG. 10D shows a vector containing a GSK3 ligand gene construct usefulfor generating stable cell lines.

The invention also encompasses polynucleotides comprising nucleotidesequences encoding ligands, homopolyligands, and heteropolyligands. Thepolynucleotides of the invention are optionally linked to additionalnucleotide sequences encoding epitopes, reporters and/or localizationsignals. Further, the nucleic acids of the invention are optionallyincorporated into vector polynucleotides. The polynucleotides areoptionally flanked by nucleotide sequences comprising restrictionendonuclease sites and other nucleotides needed for restrictionendonuclese activity. The flanking sequences optionally provide cloningsites within a vector. The restriction sites can include, but are notlimited to, any of the commonly used sites in most commerciallyavailable cloning vectors. Examples of such sites are those recognizedby BamHI, ClaI, EcoRI, EcoRV, SpeI, AflII, NdeI, NheI, XbaI, XhoI, SphI,NaeI, SexAI, HindIII, HpaI, and PstI restriction endonucleases. Sitesfor cleavage by other restriction enzymes, including homingendonucleases, are also used for this purpose. The polynucleotideflanking sequences also optionally provide directionality of subsequencecloning. It is preferred that 5′ and 3′ restriction endonuclease sitesdiffer from each other so that double-stranded DNA can be directionallycloned into corresponding complementary sites of a cloning vector.

Ligands and polyligands with or without localization signals, epitopesor reporters are alternatively synthesized by recombinant techniques.Polynucleotide expression constructs are made containing desiredcomponents and inserted into an expression vector. The expression vectoris then transfected into cells and the polypeptide products areexpressed and isolated. Ligands made according to recombinant DNAtechniques have utility as research tools and/or therapeutics.

The following is an example of how polynucleotides encoding ligands andpolyligands are produced. Complimentary oligonucleotides encoding theligands and flanking sequences are synthesized and annealled. Theresulting double-stranded DNA molecule is inserted into a cloning vectorusing techniques known in the art. When the ligands and polyligands areplaced in-frame adjacent to sequences within a transgenic gene constructthat is translated into a protein product, they form part of a fusionprotein when expressed in cells or transgenic animals.

Another embodiment of the invention relates to selective control oftransgene expression in a desired cell or organism. The promotor portionof the recombinant gene can be a constitutive promotor, anon-constitutive promotor, a tissue-specific promotor (constitutive ornon-constitutive) or a selectively controlled promotor. Differentselectively controlled promotors are controlled by different mechanisms.RheoSwitch^(R) is an inducible promotor system available from RheoGene.Temperature sensitive promotors can also be used to increase or decreasegene expression. An embodiment of the invention comprises a ligand orpolyligand gene construct whose expression is controlled by an induciblepromotor. In one embodiment, the inducible promotor is tetracyclineinducible.

Polyligands are modular in nature. An aspect of the instant invention isthe combinatorial modularity of the disclosed polyligands. Anotheraspect of the invention are methods of making these modular polyligandseasily and conveniently. In this regard, an embodiment of the inventioncomprises methods of modular subsequence cloning of genetic expressioncomponents. When the ligands, homopolyligands, heteropolyligands andoptional amino acid expression components are synthesized recombinantly,one can consider each clonable element as a module. For speed andconvenience of cloning, it is desirable to make modular elements thatare compatible at cohesive ends and are easy to insert and clonesequentially. This is accomplished by exploiting the natural propertiesof restriction endonuclease site recognition and cleavage. One aspect ofthe invention encompasses module flanking sequences that, at one end ofthe module, are utilized for restriction enzyme digestion once, and atthe other end, utilized for restriction enzyme digestion as many timesas desired. In other words, a restriction site at one end of the moduleis utilized and destroyed in order to effect sequential cloning ofmodular elements. An example of restriction sites flanking a codingregion module are sequences recognized by the restriction enzymes NgoMIV and Cla I; or Xma I and Cla I. Cutting a first circular DNA with NgoMIV and Cla I to yield linear DNA with a 5′ NgoM IV overhang and a 3′ ClaI overhang; and cutting a second circular DNA with Xma I and Cla I toyield linear DNA with a 5′Cla I overhang and a 3′ Xma I overhanggenerates first and second DNA fragments with compatible cohesive ends.When these first and second DNA fragments are mixed together, annealed,and ligated to form a third circular DNA fragment, the NgoM IV site thatwas in the first DNA and the Xma I site that was in the second DNA aredestroyed in the third circular DNA. Now this vestigial region of DNA isprotected from further Xma I or NgoM IV digestion, but flankingsequences remaining in the third circular DNA still contain intact 5′NgoM IV and 3′Cla I sites. This process can be repeated numerous timesto achieve directional, sequential, modular cloning events. Restrictionsites recognized by NgoM IV, Xma I, and Cla I endonucleases represent agroup of sites that permit sequential cloning when used as flankingsequences.

Another way to assemble coding region modules directionally andsequentially employs linear DNA in addition to circular DNA. Forexample, like the sequential cloning process described above,restriction sites flanking a coding region module are sequencesrecognized by the restriction enzymes NgoM IV and Cla I; or Xma I andCla I. A first circular DNA is cut with NgoM IV and Cla I to yieldlinear DNA with a 5′ NgoM IV overhang and a 3′Cla I overhang. A secondlinear double-stranded DNA is generated by PCR amplification or bysynthesizing and annealing complimentary oligonucleotides. The secondlinear DNA has 5′Cla I overhang and a 3′ Xma I overhang, which arecompatible cohesive ends with the first DNA linearized. When these firstand second DNA fragments are mixed together, annealed, and ligated toform a third circular DNA fragment, the NgoM IV site that was in thefirst DNA and the Xma I site that was in the second DNA are destroyed inthe third circular DNA. Flanking sequences remaining in the thirdcircular DNA still contain intact 5′ NgoM IV and 3′Cla I sites. Thisprocess can be repeated numerous times to achieve directional,sequential, modular cloning events. Restriction sites recognized by NgoMIV, Xma I, and Cla I endonucleases represent a group of sites thatpermit sequential cloning when used as flanking sequences. This processis depicted in FIG. 11.

One of ordinary skill in the art recognizes that other restriction sitegroups can accomplish sequential, directional cloning as describedherein. Preferred criteria for restriction endonuclease selection areselecting a pair of endonucleases that generate compatible cohesive endsbut whose sites are destroyed upon ligation with each other. Anothercriteria is to select a third endouclease site that does not generatesticky ends compatible with either of the first two. When such criteriaare utilized as a system for sequential, directional cloning, ligands,polyligands and other coding regions or expression components can becombinatorially assembled as desired. The same sequential process can beutilized for epitope, reporter, and/or localization signals.

Polyligands and methods of making polyligands that modulate GSK3activity are disclosed. Therapeutics include delivery of purified ligandor polyligand with or without a localization signal to a cell.Alternatively, ligands and polyligands with or without a localizationsignals are delivered via adenovirus, lentivirus, adeno-associatedvirus, or other viral constructs that express protein product in a cell.

Methods

Assays. Ligands of the invention are assayed for kinase modulatingactivity using one or more of the following methods.

Method 1. A biochemical assay is performed employingcommercially-obtained kinase, commercially-obtained substrate,commercially-obtained kinase inhibitor (control), and semi-purifiedinhibitor ligand of the invention (decoy ligand). Decoy ligands arelinked to an epitope tag at one end of the polypeptide for purificationand/or immobilization, for example, on a microtiter plate. The taggeddecoy ligand is made using an in vitro transcription/translation systemsuch as a reticulocyte lysate system well known in the art. A vectorpolynucleotide comprising a promotor, such as T7 and/or T3 and/or SP6promotor, a decoy ligand coding sequence, and an epitope tag codingsequence is employed to synthesize the tagged decoy ligand in an invitro transcription/translation system. In vitrotranscription/translation protocols are disclosed in reference manualssuch as: Current Protocols in Molecular Biology (eds. Ausubel et al.,Wiley, 2004 edition.) and Molecular Cloning: A Laboratory Manual(Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001, thirdedition). Immunoreagent-containing methods such as western blots,elisas, and immunoprecipitations are performed as described in: UsingAntibodies: A Laboratory Manual (Harlow and Lane Cold Spring HarborLaboratory Press, 1999).

Specifically, tagged decoy ligand synthesized using an in vitrotranscription/translation system is semi-purified and added to amicrotiter plate containing kinase enzyme and substrate immobilized byan anti-substrate specific antibody. Microtiter plates are rinsed tosubstantially remove non-immobilized components. Kinase activity is adirect measure of the phosphorylation of substrate by kinase employing aphospho-substrate specific secondary antibody conjugated to horseradishperoxidase (HRP) followed by the addition of3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. The catalysisof TMB by HRP results in a blue color that changes to yellow uponaddition of phosphoric or sulfuric acid with a maximum absorbance at 450nm. The Control experiments include absence of kinase enzyme, and/orabsence of decoy ligand, and/or presence/absence of known kinaseinhibitors. A known kinase inhibitor useful in the assay isstaurosporine.

Method 2. A similar assay is performed employing the same reagents asabove but the substrate is biotinylated and immobilized by binding to astreptavidin-coated plate.

Method 3. A biochemical assay is performed employingcommercially-obtained kinase, commercially-obtained substrate,commercially-obtained kinase inhibitor (control), and semi-purifiedinhibitor ligand of the invention (decoy ligand) in a microtiter plate.A luminescent-based detection system, such as Promega's Kinase-Glo, isthen added to inversely measure kinase activity.

Specifically, tagged decoy ligand synthesized using an in vitrotranscription/translation system is semi-purified and added to amicrotiter plate containing kinase enzyme and substrate. After thekinase assay is performed, luciferase and luciferin are added to thereaction. Luciferase utilizes any remaining ATP not used by the kinaseto catalyze luciferin. The luciferase reaction results in the productionof light which is inversely related to kinase activity. Controlexperiments include absence of kinase enzyme, and/or absence of decoyligand, and/or presence/absence of known kinase inhibitors. A knownkinase inhibitor useful in the assay is staurosporine.

Method 4. A similar cell-based assay is performed employing samereagents as above, but synthesizing the decoy ligand in a mammalian cellsystem instead of an in vitro transcription/translation system. Decoyligands are linked to an epitope tag at one end of the polypeptide forimmobilization and/or for purification and/or for identification in awestern blot. Optionally, tagged decoy ligands are also linked to acellular localization signal for phenotypic comparison of pan-cellularand localized kinase modulation. A vector polynucleotide comprising aconstitutive promotor, such as the CMV promotor, a decoy ligand codingsequence, an epitope tag coding sequence, and optionally a localizationsignal coding sequence is employed to express the decoy ligand in cells.Transfection and expression protocols are disclosed in reference manualssuch as: Current Protocols in Molecular Biology (eds. Ausubel et al.,Wiley, 2004 edition.) and Molecular Cloning: A Laboratory Manual(Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001, thirdedition). Western Blots and immunoreagent-containing methods areperformed as described in: Using Antibodies: A Laboratory Manual (Harlowand Lane Cold Spring Harbor Laboratory Press, 1999).

EXAMPLES Example 1

A polypeptide comprising a heteropolyligand, an endoplasmic reticulumcellular localization signal, and a His6 epitope is synthesized.Examples of such polypeptides are generically represented by FIGS. 8A,8B, 8D, 8E and 8F. The polypeptide is synthesized on an automatedpeptide synthesizer or is recombinantly expressed and purified. Purifiedpolypeptide is solubilized in media and added to cells. The polypeptideis endocytosed by the cells, and transported to the endoplasmicreticulum. Verification is performed by immunohistochemical stainingusing an anti-His6 antibody.

Example 2

A transgene is constructed using a human cytomegalovirus (CMV) promoterto direct expression of a fusion protein comprising SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:74 (POLYLIGAND), green fluorescent protein (REPORTER),and a plasma membrane localization signal (LOCALIZATION SIGNAL). Such atransgene is generically represented by FIG. 9C. The transgene istransfected into cells for transient expression. Verification ofexpression and location is performed by visualization of greenfluorescent protein (GFP) by confocal microscopy.

Example 3

A transgene construct is built to produce a protein product withexpression driven by a tissue-specific promoter. The transgene comprisesa synthetic gene expression unit engineered to encode three domains.Each of these three domains is synthesized as a pair of complimentarypolynucleotides that are annealed in solution, ligated and inserted intoa vector. Starting at the amino-terminus, the three domains in theexpression unit are nucleotide sequences that encode a GSK3 ligand, aFLAG™ epitope, and a nuclear localization signal. The GSK3 ligand is amonomeric ligand, homopolymeric ligand or heteropolymeric ligand asdescribed herein. Nucleotide sequences encoding a FLAG™ epitope areplaced downstream of nucleotide sequences encoding the GSK3 ligand.Finally, nucleotide sequences encoding the localization signal areplaced downstream of those encoding the FLAG™ epitope. The assembledgene expression unit is subsequently subcloned into an expressionvector, such as that shown in FIG. 10A, and used to transientlytransfect cells. Verification is performed by immunohistochemicalstaining using an anti-FLAG™ antibody.

Example 4

Modulation of GSK3 cellular function by subcellularly localized GSK3polyligand is illustrated. A transgene construct containing nucleicacids that encode a polyligand fusion protein, epitope, and nuclearlocalization signal is made. The expression unit contains nucleotidesthat encode SEQ ID NO: 1 (POLYLIGAND), a c-Myc epitope (EPITOPE), and anuclear localization signal (LOCALIZATION SIGNAL). This expression unitis subsequently subcloned into a vector between a CMV promoter and anSV40 polyadenylation signal (Generically depicted in FIG. 10A). Thecompleted transgene-containing expression vector is then used totransfect cells. Inhibition of GSK3 activity is demonstrated bymeasuring phosphorylation of endogenous substrates against controls.

Example 5

Ligand function and localization is demonstrated in vivo by making atransgene construct used to generate mice expressing a ligand fusionprotein targeted to the endoplasmic reticulum. The transgene constructis shown generically in FIG. 10B. The expression unit containsnucleotides that encode a tetramer of SEQ ID NO: 112, a hemagluttininepitope, and a nuclear localization signal. This expression unit issubsequently subcloned into a vector between nucleotide sequencesincluding an inducible promoter and an SV40 polyadenylation signal. Thecompleted transgene is then injected into pronuclei of fertilized mouseoocytes. The resultant pups are screened for the presence of thetransgene by PCR. Transgenic founder mice are bred with wild-type mice.Heterozygous transgenic animals from at least the third generation areused for the following tests, with their non-transgenic littermatesserving as controls.

Test 1: Southern blotting analysis is performed to determine the copynumber. Southern blots are hybridized with a radio-labeled probegenerated from a fragment of the transgene. The probe detects bandscontaining DNA from transgenic mice, but does not detect bandscontaining DNA from non-transgenic mice. Intensities of the transgenicmice bands are measured and compared with the transgene plasmid controlbands to estimate copy number. This demonstrates that mice in Example 5harbor the transgene in their genomes.

Test 2: Tissue homogenates are prepared for Western blot analysis. Thisexperiment demonstrates the transgene is expressed in tissues oftransgenic mice because hemagluttinin epitope is detected in transgenichomogenates but not in non-transgenic homogenates.

Test 3: Function is assessed by phenotypic observation or analysisagainst controls.

These examples demonstrate delivery of ligands to a localized region ofa cell for therapeutic or experimental purposes. The purifiedpolypeptide ligands can be formulated for oral or parenteraladministration, topical administration, or in tablet, capsule, or liquidform, intranasal or inhaled aerosol, subcutaneous, intramuscular,intraperitoneal, or other injection; intravenous instillation; or anyother routes of administration. Furthermore, the nucleotide sequencesencoding the ligands permit incorporation into a vector designed todeliver and express a gene product in a cell. Such vectors includeplasmids, cosmids, artificial chromosomes, and modified viruses.Delivery to eukaryotic cells can be accomplished in vivo or ex vivo. Exvivo delivery methods include isolation of the intended recipient'scells or donor cells and delivery of the vector to those cells, followedby treatment of the recipient with the cells.

Disclosed are ligands and polyligands that modulate GSK3 activity andmethods of making and using these ligands. The ligands and polyligandsare synthesized chemically or recombinantly and are utilized as researchtools or as therapeutics. The invention includes linking the ligands andpolyligands to cellular localization signals for subcellulartherapeutics.

1. An isolated polypeptide heteropolyligand, wherein theheteropolyligand modulates GSK3 activity.
 2. The isolated polypeptide ofclaim 1, comprising an amino acid sequence at least 80% identical to SEQID NO:4 or SEQ ID NO:7 or SEQ ID NO:10.
 3. An isolated fusionpolypeptide comprising two or more polypeptides selected from SEQ IDNOS:46-122, wherein Xaa is any amino acid.
 4. The isolated fusionpolypeptide of claim 2, wherein at least one amino acid designated asXaa is an amino acid other than serine or threonine.
 5. The isolatedfusion polypeptide of claim 2, wherein the fusion polypeptide comprisestwo or more polypeptides selected from SEQ ID NOS:46-111.
 6. Theisolated fusion polypeptide of claim 2, wherein the fusion polypeptidecomprises two or more polypeptides selected from SEQ ID NOS: 112-122. 7.An isolated polypeptide homopolyligand, wherein the homopolyligandmodulates GSK3 activity.
 8. The isolated polypeptide homopolyligand ofclaim 7, wherein the homopolyligand comprises monomers selected from thegroup consisting of SEQ ID NOS:46-122, wherein Xaa is any amino acid. 9.The isolated polypeptide homopolyligand of claim 7, comprising SEQ IDNO:
 1. 10. The heteropolyligand of claim 1 linked to one or more of: alocalization signal, an epitope tag, a reporter.
 11. An isolatedpolynucleotide comprising a nucleotide sequence encoding the polypeptideof claim
 1. 12. A vector comprising a polynucleotide of claim
 10. 13. Ahost cell comprising a polynucleotide claim
 10. 14. A non-human organismcomprising a polynucleotide of claim
 10. 15. The polynucleotide of claim10 operably linked to a promoter.
 16. The polynucleotide operably linkedto a promoter of claim 14, wherein the promoter is an induciblepromoter.
 17. The isolated polynucleotide of claim 10, wherein thepolynucleotide is flanked on one end by a sequence cleavable by NgoM IV,and wherein the polynucleotide is flanked on the other end by sequencescleavable by Xma I and Cla I.
 18. The isolated polynucleotide of claim10, wherein the polynucleotide comprises SEQ ID NO:5 or SEQ ID NO:6 orSEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:12.
 19. A methodof inhibiting GSK3 in a cell comprising transfecting a vector of claim12 into a host cell and culturing the transfected host cell underconditions suitable to produce at least one copy of the polypeptide.